The main focus of the proposed work in this revised application is to develop an understanding of how the HIV-1 Gag polyprotein is trafficked through the cell to the plasma membrane during virion assembly, and how the Gag cleavage product p17 MA is released from the plasma membrane for transport to the nucleus during infection. Central to both of these questions is the role of N-terminal myristylation. The experimental approach for the first aim is to determine the specific sequences within p17 MA that are required to direct plasma membrane targeting to a heterologous protein. The experimental approach in aim two is to follow the trafficking pathway of Gag using pulse/chase, subcellular fractionation, and protein complex analysis. The experimental approach for aim three is to determine the role of Gag proteolytic cleavage and phosphorylation in the release of p17 MA from the membrane to allow nuclear localization of the preintegration complex after infection.